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目的 建立缺氧诱导体外培养的成人心肌细胞凋亡情况。方法 用胰蛋白酶和胶原酶进行消化 ,用差速黏附贴壁法、Brdu加入和人为破坏成纤维细胞法纯化心肌细胞 ,用IMDM培养基培养 ,4～ 6d后将体外培养的成人心肌细胞置于 3 %氧气、92 %氮气及 5 %二氧化碳孵箱中 ,6、12、2 4、48h缺氧后再恢复正常条件培养 4h ,造成缺氧损伤所致细胞凋亡模型 ,分别用倒置显微镜、电镜、TUNEL法技术检测凋亡发生的情况。结果 心肌细胞经历缺氧损伤后 ,倒置显微镜、电镜、TUNEL染色均观察到凋亡阳性细胞。TUNEL法定量检测 ,心肌细胞缺氧培养 6、12、2 4、48h后 ,其凋亡率分别为 :( 3 3± 0 9) %、( 8 3± 1 8) %、( 16 1± 2 6) %和( 19 4± 2 3 ) %。结论 心肌细胞凋亡率随着缺氧时间的延长而增高 ,认为缺氧一定时间可以诱导体外培养的成人心肌细胞凋亡
Objective To establish hypoxia-induced apoptosis of adult cardiomyocytes in vitro. Methods The cells were digested with trypsin and collagenase. Cardiomyocytes were purified by differential adhesion adherent method, Brdu addition and human destruction of fibroblasts, and cultured in IMDM medium. After 4 to 6 days, adult cardiomyocytes cultured in vitro were placed 3% oxygen, 92% nitrogen and 5% carbon dioxide. After 6, 12, 2, and 48 hours hypoxia, the cells were returned to normal condition and cultured for 4 hours. The apoptosis models induced by hypoxia injury were induced by inverted microscope and electron microscope , TUNEL method to detect the occurrence of apoptosis. Results After myocardial cells underwent hypoxic injury, apoptotic cells were observed by inverted microscope, electron microscope and TUNEL staining. TUNEL assay showed that the apoptotic rates of cardiomyocytes were (3 3 ± 0 9)%, (8 3 ± 1 8)%, (16 1 ± 2) 6)% and (19 4 ± 2 3)%. Conclusions The apoptosis rate of cardiomyocytes is increased with the prolongation of hypoxia. It is suggested that hypoxia can induce the apoptosis of cultured cardiomyocytes in vitro